Recent observations of folate pool dynamics in cells treated with trimetrexate or methotrexate have revealed different folate depletion responses in breast and colon cancer cell lines than in hepatoma lines. An explanation of this difference is being sought in terms of the biochemical kinetics of the folate cycle. A folate cycle biochemical network model has been employed to predict that the enzyme activity differences known to exist across several colon cancer lines are sufficient to cause the diverse patterns of folate depletion observed. Cells with both ten- and fortyfold elevations in thymidylate synthase (TS) activity were predicted to exhibit nearly complete folate depletion when exposed to 1-mu(M) doses of methotrexate (MTX). Experiments have been completed in the Medicine Branch, NCI, on two cell lines selected for these increased levels of activity (plus controls) in which the cells were exposed to both 1-mu(M) and 10-mu(M) doses of MTX. Comparisons of the human colon line data with theoretical estimates based largely on human breast line parameters showed agreement at all nonzero doses and TS activities. Agreement, however, was lacking in the cell line with normal TS activity exposed to the higher MTX dose; theory predicted a short-term increase in 10-formyl tetrahydrofolate, while experimentation showed a rapid decrease. Possibly, this disparity is due to an overrepresentation of the sensitivity of TS to MTX polyglutamate inhibition, an effect more noticeable at larger drug doses.